A specialized population of cells known as podocytes plays a crucial and dynamic role in establishing and maintaining the glomerular filtration barrier. Indeed, mutations in three podocyte proteins that will be studied in this research - TRPC6, nephrin, and Neph1- lead to severe proteinuria. However, the cellular physiology of podocytes has not been extensively studied. Diabetic nephropathy is the leading cause of kidney failure in the developed world. The earliest sign of this condition is excretion of minute amounts of albumin in the urine (microalbuminuria), but over a period of years the condition progresses to end- stage renal disease, at least in a subpopulation of patients. Podocytes are affected in the earliest stages of diabetic nephropathy and are direct targets for insulin action. However, the mechanisms whereby insulin regulates the functional properties of podocytes are largely unexplored. Preliminary data in this proposal show that insulin modulates two classes of cation channels known to play a major role in regulating intracellular Ca2+ dynamics - the Ca2+ permeable cation channels of the TRPC family, especially TRPC6;and large-conductance (BKCa-type) Ca2+-activated K+ channels encoded by the Slo1 gene. Both classes of channels are functionally significant in podocytes, and there are cogent biophysical reasons to expect them to be coordinately regulated in non-excitable cells such as podocytes. We have also shown that TRPC6 and BKCa channels bind to the podocyte scaffolding protein nephrin, and that insulin and nephrin modulate the trafficking of BKCa channels in podocytes and in heterologous expression systems. BKCa channels also bind to Neph1. In this revised application, we have also shown that culturing podocytes in high glucose causes marked deficits in BKCa trafficking in podocytes that appear to be accompanied by cytoskeletal alterations. This research will use electrophysiological, biochemical, and molecular approaches to examine the mechanisms whereby insulin stimulates current through BKCa channels (Specific Aim 1) and TRPC channels (Specific Aim 2), and we will examine how channel function is altered in cell and animal models of diabetes (Specific Aim 3). The overall hypothesis of this research is that insulin acutely modulates the gating properties and trafficking of channels in part through actions on the cytoskeleton that are sensitive to high glucose and other conditions in diabetes. By examining acute and sustained effects of insulin on the cellular physiology of podocytes, we will provide information necessary for an understanding of what happens when insulin signaling is altered. The results may suggest strategies for pharmacological intervention. PUBLIC HEALTH RELEVANCE: This project will examine the effects of insulin on a population of cells in the kidney called podocytes, which are essential components of the filtration barrier that prevents proteins and other macromolecules from entering the urine. Podocytes are affected in the earliest stages of diabetic kidney disease, the leading cause of kidney failure in the developed world. Practically nothing is currently known about the effects of insulin on podocytes, which limits what we can know about diabetic nephropathy, especially in its earliest stages.